Lipase of Mucor pusillus

Lipase of Mucor pusillus NRRL 2543 was recovered with ammonium sulfate precipitation, gel filtration on Sephadex G-75, and anion-exchange chromatography on diethylaminoethyl-Sephadex A-50. Maximal glycerol ester hydrolase (lipase) activity was observed at pH 5.0 to 5.5 and 50 C when trioctanoin and olive oil were used as substrates. The enzyme also showed esterase activity; it hydrolyzed, with the exception of methyl butyrate, all methyl esters tested. A minimum chain length of six carbons appeared to be a requirement for esterase activity, which was maximal at about pH 5.5 with methyl dodecanoate (C(12)) as the substrate. Neither the glycerol ester hydrolase (lipase) nor the esterase activity of the enzyme appeared to be affected by thiol group inhibitors, chelating agents, and reducing compounds. On the other hand, hydrolysis of triolein and methyl dodecanoate was arrested to the same extent in the presence of diisopropyl fluorophosphate, which suggested the involvement of serine in the active center of the enzyme. The enzyme remained stable during a 30-day storage at - 10 C.     

Purification and partial amino acid sequences of an esterase from tomato

Screening of 18 suspension plant cell cultures of taxonomically distant species revealed that a methyl jasmonate hydrolysing enzyme activity (0.21-5.67 pkat/mg) occurs in all species so far analysed. The methyl jasmonate hydrolysing esterase was purified from cell cultures of Lycopersicon esculentum using a five-step procedure including anion-exchange chromatography, gel-filtration and chromatography on hydroxylapatite. The esterase was purified 767-fold to give an almost homogenous protein in a yield of 2.2%. The native enzyme exhibited a M(r) of 26 kDa (gel-filtration chromatography), which was similar to the M(r) determined by SDS-PAGE and MALDI-TOF analysis (M(r) of 28547 kDa). Enzyme kinetics revealed a K(m) value of 15 microM and a V(max) value of 7.97 nkat/mg, an pH optimum of 9.0 and a temperature optimum of 40 degrees C. The enzyme also efficiently hydrolyzed methyl esters of abscisic acid, indole-3-acetic acid, and fatty acids. In contrast, methyl esters of salicylic acid, benzoic acid and cinnamic acid were only poor substrates for the enzyme. N-Methylmaleimide, iodacetamide, bestatin and pepstatin (inhibitors of thiol-, metal- and carboxyproteases, respectively) did not inactivate the enzyme while a serine protease inhibitor, phenylmethylsulfonyl fluoride, at a concentration of 5 mM led to irreversible and complete inhibition of enzyme activity. Proteolysis of the pure enzyme with endoproteinase LysC revealed three peptide fragments with 11-14 amino acids. N-Terminal sequencing yielded an additional peptide fragment with 10 amino acids. Sequence alignment of these fragments showed high homologies to certain plant esterases and hydroxynitrile lyases that belong to the alpha/beta hydrolase fold protein superfamily.     

Lipase of Mucor pusillus

Lipase of Mucor pusillus NRRL 2543 was recovered with ammonium sulfate precipitation, gel filtration on Sephadex G-75, and anion-exchange chromatography on diethylaminoethyl-Sephadex A-50. Maximal glycerol ester hydrolase (lipase) activity was observed at pH 5.0 to 5.5 and 50 C when trioctanoin and olive oil were used as substrates. The enzyme also showed esterase activity; it hydrolyzed, with the exception of methyl butyrate, all methyl esters tested. A minimum chain length of six carbons appeared to be a requirement for esterase activity, which was maximal at about pH 5.5 with methyl dodecanoate (C₁₂) as the substrate. Neither the glycerol ester hydrolase (lipase) nor the esterase activity of the enzyme appeared to be affected by thiol group inhibitors, chelating agents, and reducing compounds. On the other hand, hydrolysis of triolein and methyl dodecanoate was arrested to the same extent in the presence of diisopropyl fluorophosphate, which suggested the involvement of serine in the active center of the enzyme. The enzyme remained stable during a 30-day storage at - 10 C.     

Beef liver esterase. II. Kinetic properties

The kinetic parameters, k_cat and K_M, in beef liver esterase-catalyzed hydrolysis were determined for about 100 substrates, which can be classified in several groups: (1) In the ethyl ester series of fatty acids K_M decreases with elongation of the acid, while k_cat has a maximum value with pentanoate. (2) Alkyl acetates are better substrates as the alkyl moiety is longer, whereas esters with branched alkyl groups become worse substrates. (3) Aryl esters are very good substrates. (4) Esters of dicarboxylic acids are good substrates, but only one ester group is cleaved by the enzyme. Fumarate diester is susceptible to esterase hydrolysis, while maleate is not. (5) Esters of hydrophobic amino acids are very good substrates; the enzyme is not stereoselective and both the l and d stereoisomers are readily hydrolyzed. Branching at the β-carbon atom leads to loss of activity, and blocking of the amino group abolishes it. Fluoride ion and dl-malate esters are potent competitive inhibitors of the enzymic reaction. The optimal pH was found to lie between 8 and 8.5. The reaction rate increased between 5 and 40 °C then dropped sharply. The activity decreased at high salt concentration.     

Characterization of a novel lipolytic enzyme from Aspergillus oryzae

In this study, we report the characterization of a protein from Aspergillus oryzae, exhibiting sequence identity with paraben esterase from the genus Aspergillus. The coding region of 1,586 bp, including a 77-bp intron, encoded a protein of 502 amino acids. The gene without the signal peptide of 19 amino acids was cloned into a vector, pPICZαC, and expressed successfully in Pichia pastoris as an active extracellular protein. The purified recombinant protein had pH and temperature optima of 7.0–8.0 and 30 °C, respectively, and was stable at the pH range of 7.0–10.0 and up to 40 °C. The optimal substrate for hydrolysis by the purified recombinant protein, among a panel of α-naphthyl esters (C2–C16), was α-naphthyl butyrate (C4), with activity of 0.16 units/mg protein. The considerable hydrolytic activity of the purified recombinant enzyme toward tributyrin was determined. However, no paraben esterase activity was detected toward the ethyl, propyl, and butyl esters of 4-hydroxybenzoic acid. In addition, no activity was detected toward the methyl esters of ferulic, p-coumaric, caffeic, and sinapic acids that would indicate feruloyl esterase activity.     

Rupture of rat liver lysosomes mediated by l-amino acid esters

Treatment of rat liver lysosome suspensions with 0.5–20 mM α-l-amino acid esters results in a progressive loss of latency of lysosomal enzyme activity. The increase in available acid phosphatase activity in lysosomal suspensions is correlated with the decrease of turbidity of these suspensions. Ester mediated turbidity decrease is dependent upon ester concentration, and the pH and ionic strength of the suspending medium. d-Stereoisomers of amino acid esters do not exhibit comparable capacity to damage lysosomes.α-l-Amino acid esters were found to be substrates for neutral lysosomal esterase and transpeptidase activity. The d-stereoisomers are degraded at much lower rates. These data support the hypothesis that ester dependent lysosomal rupture is mediated by the specific interaction of the ester with a structural or functional lysosomal protein.     

Active site of the enzyme which demethylates receptors during bacterial chemotaxis

The CheB methylesterase catalyzes the hydrolysis of glutamyl methyl esters in bacterial chemoreceptor proteins. Studies with residue-specific inhibitors suggest that a cysteine residue is required. The nucleotide sequence of the cheB gene predicts a 349-amino acid protein with cysteine residues at positions 207 and 309. Oligonucleotide-directed mutagenesis was used to change each cysteine to an alanine. Whereas the Cys207-Ala mutation had essentially no effect on esterase activity, the Cys309-Ala mutation caused a complete inactivation of the enzyme. Cys309 is located adjacent to a sequence of amino acids which is characteristic of the beta-alpha-beta motif found in a number of nucleotide binding proteins associated with receptor function in vertebrate tissues. A central feature of this structure is Gly-X-Gly-X-X-Gly. Mutation of the second glycine in this region (Gly284) to a valine also caused a complete loss of esterase activity.     

12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoic acid: biological recognition by cholesterol esterase and acyl-CoA:cholesterol O-acyltransferase

Potential probes of protein cholesterol and fatty acid binding sites, namely, 12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoate (IFA) and its coenzyme A (IFA:CoA) and cholesteryl (IFA:CEA) esters, were synthesized. These radioactive, photoreactive lipid analogues were recognized as substrates and inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT) and cholesterol esterase, neutral lipid binding enzymes which are key elements in the regulation of cellular cholesterol metabolism. In the dark, IFA reversibly inhibited cholesteryl [14C]oleate hydrolysis by purified bovine pancreatic cholesterol esterase with an apparent Ki of 150 microM. Cholesterol esterase inhibition by IFA became irreversible after photolysis with UV light and oleic acid (1 mM) provided 50% protection against inactivation. Incubation of homogeneous bovine pancreatic cholesterol esterase with IFA:CEA resulted in its hydrolysis to IFA and cholesterol, indicating recognition of IFA:CEA as a substrate by cholesterol esterase. The coenzyme A ester, IFA:CoA, was a reversible inhibitor of microsomal ACAT activity under dark conditions (apparent Ki = 20 microM), and photolysis resulted in irreversible inhibition of enzyme activity with 87% efficiency. IFA:CoA was also recognized as a substrate by both liver and aortic microsomal ACATs, with resultant synthesis of 125IFA:CEA. IFA and its derivatives, IFA:CEA and IFA:CoA, are thus inhibitors and substrates for cholesterol esterase and ACAT. Biological recognition of these photoaffinity lipid analogues will facilitate the identification and structural analysis of hitherto uncharacterized protein lipid binding sites.     

Neutral and acid retinyl ester hydrolases associated with rat liver microsomes: relationships to microsomal cholesteryl ester hydrolases

We recently reported the presence of a neutral, bile salt-independent retinyl ester hydrolase (REH) activity in rat liver microsomes and showed that it was distinct from the previously studied bile salt-dependent REH and from nonspecific carboxylesterases (Harrison, E. H., and M. Z. Gad. 1989. J. Biol. Chem. 264: 17142-17147). We have now further characterized the hydrolysis of retinyl esters by liver microsomes and have compared the observed activities with those catalyzing the hydrolysis of cholesteryl esters. Microsomes and microsomal subfractions enriched in plasma membranes and endosomes catalyze the hydrolysis of retinyl esters at both neutral and acid pH. The acid and neutral REH enzyme activities can be distinguished from one another on the basis of selective inhibition by metal ions and by irreversible, active site-directed serine esterase inhibitors. The same preparations also catalyze the hydrolysis of cholesteryl esters at both acid and neutral pH. However, the enzyme(s) responsible for the neutral REH activity can be clearly responsible for the neutral REH activity can be clearly differentiated from the neutral cholesteryl ester hydrolase(s) on the basis of differential stability, sensitivity to proteolysis, and sensitivity to active site-directed reagents. These results suggest that the neutral, bile salt-independent REH is relatively specific for the hydrolysis of retinyl esters and thus may play an important physiological role in hepatic vitamin A metabolism. In contrast to the neutral hydrolases, the activities responsible for hydrolysis of retinyl esters and cholesterol esters at acid pH are similar in their responses to the treatments mentioned above. Thus, a single microsomal acid hydrolase may catalyze the hydrolysis of both types of ester.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate specificity of an α-amino acid ester hydrolase produced by Acetobacter turbidans A.T.C.C. 9325

A partially purified preparation of an alpha-amino acid ester hydrolase was obtained from Acetobacter turbidans A.T.C.C. 9325, which catalyses synthesis of 7-(d-alpha-amino-alpha-phenylacetamido)-3-cephem-3-methyl-4- carboxylic acid (cephalexin) from methyl d-alpha-aminophenylacetate and 7-amino-3-deacetoxycephalosporanic acid. The enzyme preparation catalysed both cephalosprin synthesis from 7-amino-3-deacetoxycephalosporanic acid and suitable amino acid esters (e.g. methyl d-alpha-aminophenylacetate, l-cysteine methyl ester, glycine ethyl ester, d-alanine methyl ester, methyl dl-alpha-aminoiso-butyrate, l-serine methyl ester, d-leucine methyl ester, l-methionine methyl ester) and the hydrolysis of such esters. The substrate specificity of the enzyme preparation for the hydrolysis closely paralleled the acyl-donor specificity for cephalosporin synthesis, even to the reaction rates. Only alpha-amino acid derivatives could act as acyl donors. The hydrogen atom on the alpha-carbon atom was not always required by acyl donors. The hydrolysis rate was markedly diminished by adding 7-amino-3-deacetoxycephalosporanic acid to reaction mixtures, but no effect on the total reaction rate (the hydrolysis rate plus synthesis rate) was observed with various concentrations of 7-amino-3-deacetoxycephalosporanic acid. Both the hydrolytic and the synthetic activities of the enzyme preparation were inhibited by high concentrations of some acyl donors (e.g. methyl d-alpha-aminophenylacetate, ethyl d-alpha-aminophenylacetate). The enzyme preparation hydrolysed alpha-amino acid esters much more easily than alpha-amino acid derivatives with an acid-amide bond.     

Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment

A novel lipase was isolated from a metagenomic library of Baltic Sea sediment bacteria. Prokaryotic DNA was extracted and cloned into a copy control fosmid vector (pCC1FOS) generating a library of >7000 clones with inserts of 24-39 kb. Screening for clones expressing lipolytic activity based on the hydrolysis of tributyrin and p-nitrophenyl esters, identified 1% of the fosmids as positive. An insert of 29 kb was fragmented and subcloned. Subclones with lipolytic activity were sequenced and an open reading frame of 978 bp encoding a 35.4-kDa putative lipase/esterase h1Lip1 (DQ118648) with 54% amino acid similarity to a Pseudomonas putida esterase (BAD07370) was identified. Conserved regions, including the putative active site, GDSAG, a catalytic triad (Ser148, Glu242 and His272) and a HGG motif, were identified. The h1Lip1 lipase was over expressed, (pGEX-6P-3 vector), purified and shown to hydrolyse p-nitrophenyl esters of fatty acids with chain lengths up to C14. Hydrolysis of the triglyceride derivative 1,2-di-O-lauryl-rac-glycero-3-glutaric acid 6'-methylresorufin ester (DGGR) confirmed that h1Lip1 was a lipase. The apparent optimal temperature for h1Lip1, by hydrolysis of p-nitrophenyl butyrate, was 35 degrees C. Thermal stability analysis showed that h1Lip1 was unstable at 25 degrees C and inactivated at 40 degrees C with t1/2 <5 min.     

Modulation of human natural killer cell function by L-leucine methyl ester: monocyte-dependent depletion from human peripheral blood mononuclear cells

L-leucine methyl ester (Leu-OMe) causes lysosomal disruption and death of human monocytes (M phi). In addition, Leu-OMe removed natural killer cell (NK) activity from human peripheral mononuclear cells (PBM). Thus, a brief preincubation of PBM with Leu-OMe (greater than 1 mM) caused irreversible loss of NK function as assessed by the lysis of K562 targets. By contrast, a variety of other amino acid methyl esters, including L-glutamic dimethyl ester, L-valine methyl ester, and L-isoleucine methyl ester caused reversible inhibition of NK activity in a manner that was similar to other lysosomotropic agents such as chloroquine and ammonium chloride, but did not cause irreversible loss of all NK function. Leu-OMe appeared to cause actual removal of NK effector cells from PBM, because K562 target binding cells, Leu-11b+ lymphocytes, and OKM1+ lymphocytes were depleted. If M phi were removed from PBM before the incubation, Leu-OMe caused only reversible inhibition of NK function in a manner similar to that observed with other amino acid methyl esters. Upon the addition of freshly isolated M phi, polymorphonuclear leukocytes, or sonicates of these cells to M phi-depleted lymphocyte populations, irreversible ablation of NK function was again observed as a result of Leu-OMe exposure. After in vitro culture, M phi lost their susceptibility to Leu-OMe toxicity and the ability to mediate the irreversible deletion of NK cells resulting from Leu-OMe incubation. These results indicate that in the absence of M phi, Leu-OMe and a variety of other amino acid methyl esters are reversible inhibitors of NK function. However, Leu-OMe is unique in that it can interact with M phi or granulocytes to effect an irreversible loss of NK activity from human peripheral blood lymphocytes.     

Characterization of a thermostable esterase activity from the moderate thermophile Bacillus licheniformis

A new esterase activity from Bacillus licheniformis was characterized from an Escherichia coli recombinant strain. The protein was a single polypeptide chain with a molecular mass of 81 kDa. The optimum pH for esterase activity was 8-8.5 and it was stable in the range 7-8.5. The optimum temperature for activity was 45 degrees C and the half-life was 1 h at 64 degrees C. Maximum activity was observed on p-nitrophenyl caproate with little activity toward long-chain fatty acid esters. The enzyme had a KM of 0.52 mM for p-nitrophenyl caproate hydrolysis at pH 8 and 37 degrees C. The enzyme activity was not affected by either metal ions or sulfydryl reagents. Surprisingly, the enzyme was only slightly inhibited by PMSF. These characteristics classified the new enzyme as a thermostable esterase that shared similarities with lipases. The esterase might be useful for biotechnological applications such as ester synthesis.     

The structure and enzymic activities of the C1r and C1s subcomponents of C1, the first component of human serum complement.

The subcomponents C1r and C1s and their activated forms C-1r and C-1s were each found to have mol.wts. in dissociating solvents of about 83000. The amino acid compositions of each were similar, but there were significant differences in the monosaccharide analyses of subcomponents C1r and C1s, whether activated or not. Subcomponents C1r and C1s have only one polypeptide chain, but subcomponents C-1r and C-1s each contain two peptide chains of approx. mol.wts. 56000 ("a" chain) and 27000 ("b" chain). The amino acid analyses of the "a" chains from each activated subcomponent are similar, as are those of the "b" chains. The N-terminal amino acid sequence of 29 residues of the C-1s "a" chain was determined, but the C-1r "a" chain has blocked N-terminal amino acid. The 20 N-terminal residues of both "b" chains are similar, but not identical, and both show obvious homology with other serine proteinases. The difference in polysaccharide content of the subcomponents C-1r and C-1s is most marked in the 'b' chains. When tested on synthetic amino acid esters, subcomponent C-1r hydrolysed both lysine and tyrosine ester bonds, but subcomponent C-1r did not hydrolyse any amino acid esters tested nor any protein substrate except subcomponent C1s. The lysine esterase activity of subcomponent C1s provides a rapid and sensitive assay of the subcomponent.     

Leishmania amazonensis: uptake and hydrolysis of 3H-amino acid methyl esters by isolated amastigotes

Intracellular and isolated amastigotes of Leishmania amazonensis can be destroyed by L-amino acid methyl esters known to disrupt mammalian lysosomes. To evaluate the mechanism(s) involved in the leishmanicidal activity, we examined the uptake and hydrolysis of tritiated esters by isolated amastigotes. After incubation with the labeled compounds, parasites were recovered, were washed on filters, and their radioactivity was determined. Alternatively, amastigotes were separated from the medium by centrifugation through oil, and the radioactivity associated with free or esterified amino acids was measured after thin-layer chromatography. The results showed that the methyl esters of Trp, Leu, and Met, which are leishmanicidal, accumulated in and were rapidly hydrolysed by the amastigotes. [3H]Leu derived from [3H]Leu-OMe remained associated with the amastigotes even after a 1-hr chase in label-free medium, but the ester species was rapidly lost upon washing of the parasites. In contrast, the esters of Ile and Ala, which are not leishmanicidal, were only slowly hydrolysed, and most of the radioactivity was lost upon washing. We have previously shown that certain amino acid esters and weak bases protect Leishmania from damage by leucine methyl ester (Leu-OMe). In the present experiments, these compounds reduced, in concentration-dependent fashion, the hydrolysis of [3H]Leu-OMe and the accumulation of [3H]Leu in the amastigotes. Overall, the results indicate that, as in lysosomal disruption, leishmanicidal activity is associated with ester hydrolysis and amino acid accumulation in the parasites. The nature and location of the parasite esterolytic enzymes requires additional investigation.     

Enantioselective ester hydrolase from Sphingobacterium sp. 238C5 useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis

A novel enzyme, β-phenylalanine ester hydrolase, useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis was characterized. The enzyme purified from the cell free-extract of Sphingobacterium sp. 238C5 well hydrolyzed β-phenylalanine esters (S)-stereospecifically. Besides β-phenylalanine esters, the enzyme catalyzed the hydrolysis of several α-amino acid esters with l-stereospecificity, while the deduced 369 amino acid sequence of the enzyme exhibited homology to alkaline d-stereospecific peptide hydrolases from Bacillus strains. Escherichia coli transformant expressing the β-phenylalanine ester hydrolase gene exhibited an about 8-fold increase in specific (S)-β-phenylalanine ethyl ester hydrolysis as compared with that of Sphingobacterium sp. 238C5. The E. coli transformant showed (S)-enantiomer specific esterase activity in the reaction with a low concentration (30 mM) of β-phenylalanine ethyl ester, while it showed both esterase and transpeptidase activity in the reaction with a high concentration (170 mM) of β-phenylalanine ethyl ester and produced β-phenylalanyl-β-phenylalanine ethyl ester. This transpeptidase activity was useful for β-phenylalanine β-peptide synthesis.     

Permeation and stereospecificity of hydrolysis of peptide esters within intact lysosomes in vitro

Various dialanine ethyl esters and di- and trimethionine methyl ester diastereomers have been shown to bring about, at a concentration range of 0.5 5mM, the loss of latency of rat liver by lysosomal acid phosphatase. Concurrently an examination was made of the hydrolysis of the peptide esters by a lysosomal enzyme composite.The total hydrolysis of peptide esters involves several steps. Using a variety of siastereomers it was possible to elucidate the stereospecificity of the enzymes involved, i.e. an ester bond involving a D-amino acid residue as well as peptide bonds involving either a DD or a DL configuration in the amino terminal end and an LD configuration in a sequence of DLD are not susceptible to enzymic hydrolysis.A correlation was found between the potency of a given peptide ester in damaging lysosomal integrity and its susceptibility to hydrolysis by the lysosomal enzyme composite. It is suggested that lysosomal rupture stems from intralysosomal peptide ester hydrolysis, leading to an accumulation of zwitteron degradation products. The broad stereospecificity of the permeating species and the discrimination between trialanine and trimethionine esters lead to the suggestion that the permeation involves partition between the medium and the non-polar regions of the lysosomal membrane.     

The Hfq-like protein NrfA of the phototrophic purple bacterium Rhodobacter capsulatus controls nitrogen fixation via regulation of nifA and anfA expression

A novel esterase catalyzing regioselective hydrolysis was purified from the membrane fraction of Microbacterium sp. 7-1W, and characterized. The enzyme was solubilized with Brij 58 and purified 13.8-fold to apparent homogeneity with 2.58% overall recovery. The relative molecular mass of the native enzyme as estimated by gel filtration was more than 600,000 Da, and the subunit molecular mass was 62,000 Da. The enzyme catalyzed cleavage of the terminal ester bonds of cetraxate esters and pantothenate esters. The K(m) and V(max) values for methyl cetraxate were 0.380 mM and 7.76 micromole min(-1) mg(-1) protein, respectively. The enzyme was inhibited by serine hydrolase inhibitors.